The protein encoded by the BRM gene (SMARCA2, SNF2, SWI2) is a member of the SWI/SNF family of proteins and is highly similar to the brahma protein of Drosophila (1). Members of this family have helicase and ATPase activities and are thought to regulate transcription of certain genes by altering the chromatin structure around those genes (2). The encoded protein is part of the large ATP-dependent chromatin remodeling complex SNF/SWI, which is required for transcriptional activation of genes normally repressed by chromatin. BRM is an epigenetically suppressed anti-cancer gene, which is silenced in wide variety of solid tumors (2). Because BRM function is key for growth control, restoring its expression routinely inhibits cancer cell growth. For this reason, restoring BRM has potential as an anticancer therapeutic modality (3-4). Its expression prevents cancer development as seen in murine models system where BRM loss potentiates cancer development 10-fold (5). It is known from preliminary studies that histone deacetylase (HDAC) inhibitors are found to restore BRM expression in cancer cell lines, but not its function (2). However, BRM is involved in many pathways and required by numerous transcription factors which control development, differentiation, DNA repair, adhesion, and growth control (6). As such, the impact of inactivating BRM and/or restoring its expression goes well beyond growth control (7).

Although pan-histone deacetylase (HDAC) inhibitors are found to restore BRM expression, but not its function in cancer cell lines, specific inhibitors of either HDAC3 or HDAC9, as well as the transcription factor GATA3 and/or MEF2D, induce a functional BRM. These other constituents involved in BRM regulation may also be the molecular targets. However, since HDAC9 and GATA3 are highly overexpressed and given the limited scope of expression of HDAC9, these proteins would be preferred targets. The fact that each are highly over-expressed is akin to EGFR in lung cancer or HER2 in breast cancer. Candidate compounds that are positive on the primary screen will be re-screened using our counter-screen, where BRM has been suppressed using anti-BRM shRNA (MG2-KDM). We have found that even the most potent inducers of BRM, are blocked by at least 50% or more by this assay. Hence, false positives will yield readouts of luciferase activity that show >50% inductions, closely approximating the levels found in the primary BRM (MG213) screen and will not have inductions <50% as observed with essentially all BRM-specific inducers.

Compounds will be then be verified as positive hits by a series of assays beginning with a third confirmatory (dose response) screening. Following the dose response screening, hits will be screened by directly measuring BRM induction by qPCR since the BRM gene is controlled by transcription. Additionally, we will determine the relative specificity the hits identified by screening each for its potential to induce a number (~10) of BRM-dependent genes. Since each of these BRM-dependent genes are controlled by different signal transduction pathways, interference in one or more cellular pathways (due to lack of specificity for BRM) will be demonstrated by a lack or reduced induction of one or more of the BRM-dependent genes. Moreover, the level of induction observed for each BRM-dependent gene is an indirect marker for the potency of BRM induction.

A secondary goal of this project is to group these verified compounds based upon their relative site of action in the pathway of BRM induction. We will use a secondary assay to differentiate compounds affecting upstream and downstream sections in the regulatory pathway. A possible additional screen will then be optionally performed using cells harboring anti-HDAC2 shRNA (MGH2KD cells). Since the deacetylation of BRM is a requirement for function thus generating a luciferase signal in these reporter cells, only the compound(s) that affect higher-level regulatory genes (such as MAP kinase inhibition) will be identified with a positive result using this assay. For those compounds having an impact lower in the pathway, we will use them to determine if they inhibit BRN2, GATA3, HDAC3, MEF2D, or perhaps HDAC9, thereby assisting us in determining which motifs of the compound underlie the re-expression of BRM. After this series of screens, all hits will then undergo a series of secondary assays to determine how well they restore BRM expression and its function in BRM-deficient cell lines. The assay provider will explore these secondary MOA studies in additional collaboration beyond the scope of this CPDP.

Assay Overview:The purpose of this assay is to identify compounds that act as activators (re-activators) of BRM function. This assay relies on the fact that BRM assists the glucocorticoid receptor (GR) in activating the MMTV promoter, which is used to drive expression of a luciferase reporter gene (7). Compounds active in this assay will restore BRM function, allowing the SWI/SNF-dependent GR to induce the MMTV promoter, leading to increased luciferase expression and well luminescence. MG213 cells are incubated in the presence of 0.1 uM Dexamethasone for 24 hours prior to be plated in 1536-well plates at 600 cells/well in 5 ul media with Dexamethasone using Kalypsys dispenser. The cells are immediately pinned with 45 nL of compound, control or DMSO, spun down, and incubated overnight at 37 C. Test compounds compound are delivered to the plates containing the MG213 cells and dexamethasone using pintool. Chembridge 5306793 is included as a positive control on each plate. OneGlo luciferase reagent is used according to the manufacturer's directions. Luminescence is measured using the Viewlux plate reader. Compounds are tested in triplicate at a final nominal concentration of 9.1 uM. Note that the cell line used for this assay has been transduced with the E1A gene, which produces the viral oncoprotein, E1A. This protein sequesters Rb (and Rb2) protein thus preventing cellular growth inhibition. At lower densities, these newly modified cells have a more linear growth rate and do not lag when plated at low density. At higher densities (which typically impacts normal cell growth via contact inhibition), these cells maintain their growth rate allowing these cells to grow to higher densities. This modified cell line allows for a greater experimental range for this assay and improves its performance (consistency) as compared to the parental MG213 cells.Protocol Summary:The MG213 cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of RPMI -1640 supplemented with 5% v/v certified fetal bovine serum, 500 ug/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). One day prior to the assay, the media is changed with induction media (growth media minus Geneticin and with 0.1uM Dexamethasone).Prior to the start of the assay 600 cells in a 5 ul volume of assay media (growth media as above except without geneticin and with 2.5% FBS and 0.1uM Dexamethasone) were dispensed into each well of 1536-well tissue culture-treated microtiter plates. The assay was started immediately by dispensing 45 nL of test compound in DMSO (0.69 % final DMSO concentration), DMSO alone, or ChemBridge 5306793 (CAS 244052-79-7) to the appropriate wells. Next, the plates were incubated for 24 hours at 37 C (5% CO2, 95% RH). The assay was stopped by dispensing 5 ul of One Glo luciferase substrate to each well, followed by incubation at room temperature for 15 minutes. Well luminescence was measured on the ViewLux plate reader.The percent activation for each compound was calculated as follows:%_Activation = ( 1 - ( ( Test_Compound - Median_High_Control ) / ( Median_Low_Control - Median_High_Control ) ) ) * 100Where:Test_Compound is defined as wells containing test compound.Low_Control is defined as wells containing DMSO.High_Control is defined as wells containing ChemBridge 5306793 (CAS 244052-79-7).PubChem Activity Outcome and Score:A mathematical algorithm was used to determine nominally activating compounds in the Primary Screen. Two values were calculated for each assay plate: (1) the average percent activation of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % activation than that particular plate's cutoff parameter was declared active.The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.The PubChem Activity Score range for active compounds is 100-2, and for inactive compounds 2-0.List of Reagents:MG213 cell line (provided by Assay Provider)RPMI-1640 medium (Invitrogen, 11875-119Geneticin (Invitrogen, part 10131-035)100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)Trypsin-EDTA solution (Invitrogen, part 25200-056)Fetal Bovine Serum (Invitrogen, part 16000-044)One Glo Assay Kit (Promega, part E6130/QTE30675 )ChemBridge 5306793 (CAS 244052-79-7), ChembridgeT-175 tissue culture flasks (Corning, part 431080)1536-well plates (Corning, part 789173)

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate luciferase activity, and compounds that quench or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.